Empagliflozin-Pretreated Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Attenuated Heart Injury

Objective Small extracellular vesicles derived from mesenchymal stem cells (MSCs) play important roles in cardiac protection. Studies have shown that the cardiovascular protection of sodium-glucose cotransporter 2 inhibitor (SGLT2i) is independent of its hypoglycemic effect. This study is aimed at investigating whether small extracellular vesicles derived from MSCs pretreated with empagliflozin (EMPA) has a stronger cardioprotective function after myocardial infarction (MI) and to explore the underlying mechanisms. Methods and Results We evaluated the effects of EMPA on MSCs and the effects of EMPA-pretreated MSCs-derived small extracellular vesicles (EMPA-sEV) on myocardial apoptosis, angiogenesis, and cardiac function after MI in vitro and in vivo. The small extracellular vesicles of control MSCs (MSC-sEV) and EMPA-pretreated MSCs were extracted, respectively. Small extracellular vesicles were cocultured with apoptotic H9c2 cells induced by H2O2 or injected into the infarcted area of the Sprague-Dawley (SD) rat myocardial infarction model. EMPA increased the cell viability, migration ability, and inhibited apoptosis and senescence of MSCs. In vitro, EMPA-sEV inhibited apoptosis of H9c2 cells compared with the control group (MSC-sEV). In the SD rat model of MI, EMPA-sEV inhibited myocardial apoptosis and promoted angiogenesis in the infarct marginal areas compared with the MSC-sEV. Meanwhile, EMPA-sEV reduced infarct size and improved cardiac function. Through small extracellular vesicles (miRNA) sequencing, we found several differentially expressed miRNAs, among which miR-214-3p was significantly elevated in EMPA-sEV. Coculture of miR-214-3p high expression MSC-derived small extracellular vesicles with H9c2 cells produced similar protective effects. In addition, miR-214-3p was found to promote AKT phosphorylation in H9c2 cells. Conclusions Our data suggest that EMPA-sEV significantly improve cardiac repair after MI by inhibiting myocardial apoptosis. miR-214-3p at least partially mediated the myocardial protection of EMPA-sEV through the AKT signaling pathway.


Introduction
Acute myocardial infarction (AMI) is the major cause of morbidity and mortality all over the world [1]. Percutaneous transluminal coronary intervention (PCI), as the main treatment for acute myocardial infarction, can improve the prognosis but cannot avoid the occurrence of myocardial cell death, ventricular remodeling, and heart failure [2]. Bone marrowderived MSCs are a promising approach to the treatment of cardiac injury after myocardial infarction (MI). However, therapeutic effect on MI by MSCs is limited due to the poor survival rate given a hostile microenvironment in an ischemic heart. At the same time, some studies have shown that the beneficial effect of MSCs is mainly due to the paracrine action of secretory factors, rather than the direct muscle regeneration of MSCs or MSC derived cells [3,4].
Currently, cell-free therapy based on MSCs derived small extracellular vesicles has made remarkable progress in myocardial protection [4,5]. Small extracellular vesicles are lipid bilayered structures, 30-150 nm in size, enclosing cargo containing messenger ribonucleic acid (mRNA), miRNAs, growth factors, and proteins for transfer into recipient cells [6,7]. Small extracellular vesicles have great advantages, such as high cyclic stability, dose control of transplantation, lack of immunogenicity, low toxicity, and easy functionalization. However, the therapeutic effect of small extracellular vesicles lack modification is not as effective as expected. Therefore, small extracellular vesicles can be optimized by various engineering methods to improve the targeting and therapeutic effect of small extracellular vesicles, such as pharmacological compound-pretreated MSCs and genemodified MSCs [8]. MSCs pretreated with drugs such as atorvastatin [9], rosuvastatin [10], and curcumin [11] have been shown to improve the survival of local cardiomyocytes and promote angiogenesis. At present, small extracellular vesicles released from MSCs pretreated with atorvastatin can significantly improve cardiac function in the MI model of SD rats, and lncRNA H19 is partially involved in cardiac repair mediated by angiogenesis [12]. So, the development of new strategies to optimize cell-free therapy is expected to play an important role in future clinical applications.
EMPA, a sodium glucose cotransporter 2 inhibitor (SGLT2i), is a new oral hypoglycemic agent for the treatment of type 2 diabetes mellitus (T2DM). SGLT2i were shown to decrease mortality from cardiovascular diseases in the EMPA-REG trial [13]. Inhibition of the SGLT2 reduces cardiovascular morbidity and mortality in patients with T2DM with atherosclerotic, cardiovascular disease [14,15]. SGLT2i significantly improve cardiovascular outcomes including cardiovascular and all-cause mortality in patients with HF without an increased risk of serious adverse events [16]. Combined with the benefits of SGLT2i in cardiovascular disease and the potential benefits of biomimetic small extracellular vesicles for stem cell self-therapy [17,18], it becomes critical to determine the effects of EMPA pretreatment on the functions of MSCs and MSC-derived small extracellular vesicles.
In this study, we investigated the cardioprotective effects of EMPA-pretreated MSC-derived small extracellular vesicles in vitro and in vivo. Compared with MSC-sEV, EMPA-sEV significantly enhanced cardiac function and promoted angiogenesis. The apoptosis rate of EMPA-sEV was also significantly reduced. To explore the underlying molecular mechanisms, we found that EMPA-sEV showed significantly increased miR-214-3p expression levels. The high expression of miR-214-3p could simulate the improvement effect of EMPA-sEV. Therefore, our study suggests that EMPA preconditioning may strengthen the therapeutic effect of the small extracellular vesicles derived from MSCs on MI by inhibiting myocardial apoptosis through a paracrine mechanism. In addition, miR-214-3p appears to mediate the protective effect of EMPA-sEV on the heart after AMI.

Small Extracellular Vesicle Isolation and Characterization.
MSCs were cultured in complete medium to 80% confluence and then incubated with extracellular vesicle-free medium for 48 h. After 48 h, conditioned medium was collected and centrifuged at 1500 g for 30 min to remove apoptotic bodies and cell debris, followed by incubation with Ribo™ Exosome Isolation Reagent (for cell culture media, RiboBio, C10130-2) for 12 h at 4°C. The supernatant was centrifuged at 2000 g for 30 min. The supernatant was discarded, and the pellet was suspended in PBS (100 μl) and stored at -80°C.
The particle size and concentration of small extracellular vesicles were analyzed using nanoparticle tracking analysis (NTA). The collected small extracellular vesicles were fixed on carbon-coated copper grids with 1% glutaraldehyde and then stained with 1% phosphotungstic acid. The samples were examined using a JEM-2100 transmission electron microscope (TEM). And western blotting for CD63, CD81, and TGS101 was used to characterize the collected EMPA-sEV and MSC-sEV. To verify whether small extracellular vesicles could be absorbed by H9c2 cells, small extracellular vesicles were labeled with PHK26 and cocultured with H9c2 cells for 24 hours. And the cell nucleus were stained with DAPI after coculture. . SD rats (6-8 weeks-old; male) were procured from Animal Core Facility of Nanjing Medical University. The rats were acclimated to sterile animal management conditions for 7 days. The animals were randomly assigned to different treatment groups (n = 6). Anesthesia was performed by intraperitoneal injection of 100 mg/kg ketamine, and assisted ventilation was performed by endotracheal intubation on a small animal ventilator (ALC-V, ALCBIO). Myocardial infarction was caused by ligation of the left anterior descending coronary artery, as previously reported [20]. After MI, 50 μl PBS or small extracellular vesicles (from 1 × 10 6 cultured MSCs) in a total volume of 50 μl PBS was injected into the myocardium of each SD rat at three different locations in the infarcted area. Left ventricular function was evaluated 23 and 36 days after infarction. To avoid experimental bias and variation, the person performing the surgery is unaware of the grouping.   Oxidative Medicine and Cellular Longevity number of apoptotic cells in the tissue sections was quantified by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay.

Small Extracellular Vesicle miRNA Sequencing and
Bioinformatics Analyses. The miRNA sequencing was performed in both EMPA-sEV and MSC-sEV. Small extracellular vesicles' miRNA sequence was analyzed by RiboBio (Guangzhou, China) using the Illumina HiSeq™ 2500 instrument. Differentially expressed miRNA were identified by fold change ðjlog 2ðFoldChangeÞj > 1Þ and significance levels (P value < 0.05). Bioinformatics analysis was performed, including differential expression miRNA analysis, miRNA target gene prediction, GO analysis, and KEGG pathway enrichment analysis.
2.14. miRNA Transfection. miR-214-3p mimics (miR-214 mimics) (50 nmol/l) and negative controls (NC mimics) (50 nmol/l) were transfected using riboFECT™ CP Reagent according to the manufacturer's instructions. In short, MSCs were cultured at 50% confluence. miR-214 mimics and the NC mimics were mixed with transfection reagents and then added to cell culture medium. The transfection efficiency was determined by RT-qPCR. The transfected miRNA sequences are shown in supplementary table 1.

Statistical
Analysis. Data were shown in bar graphs as mean ± standard error of mean (SEM). Continuous variables were compared using Student's t-tests. When there were more than two groups of data, one-way analysis of variance (ANOVA) was used for analysis. Data were analyzed with GraphPad Prism 8 for Windows, version 8.01 (GraphPad Software, Inc.). Graphs were assembled in GraphPad Prism 8. A P value < 0.05 was considered significant.

EMPA Enhances Mesenchymal Stem Cell Viability.
Consistent with previous studies, MSCs rapidly adhered to tissue culture containers and took on a fibroblast-like morphology in culture [20] (Figure 1(a)). The multiple differentiation potential of human bone marrow MSCs into adipogenesis, osteogenesis, and chondrogenesis was confirmed by oil red staining, alizarin red staining, and Alcian blue staining (Figure 1(a)). MSCs were also characterized by the expression of CD44, CD73, and CD105. Meanwhile, MSCs were also negative for CD45, CD31, and CD34 ( Figure 1(b)).
To evaluate the optimal therapeutic dose for EMPA, MSCs were treated at different concentrations. The cell viability of MSCs was determined by CCK-8 assay after 24 h and 48 h culture, respectively. The data showed that MSCs exhibited better cell viability at 500 nM and 48 h compared with other concentrations and control (Supplementary figure 1). Therefore, we treated MSCs with EMPA at 500 nM concentration and cultured them for 48 h as unified experimental conditions for in vitro analysis. We evaluated the proliferation of MSCs by EdU staining. Compared with the control group, the cell proliferation ability of EMPA group was significantly increased (Figures 1(c) and 1(d)). In addition, the wound healing assay showed that the EMPA group showed higher migration compared to the control group (Figure 1(e)). As is exhibited in Figures 1(f) and 1(g), the levels of SA-β-gal activity were significantly decreased in the EMPA group compared with the control. Furthermore, in contrast to the control, the EPMA group demonstrated a reduced expression level of senescence-associated protein 21 (Figure 1(h)).
In conclusion, EMPA-pretreated MSCs showed better proliferation, stronger migration, and reduced senescence of the MSCs.   (Figure 2(a)). Similarly, the MSC-derived small extracellular vesicles in the control group also had the same structure and size (Figure 2(a)). Next, NTA was used to analyze the small extracellular vesicles' size distribution and concentration. As shown in Figures 2(b) and 2(d) and supplementary table 2, EMPA-sEV and MSC-sEV have similar particle sizes and concentrations. Western blot assay showed that there were specific marker proteins of small extracellular vesicles in both groups, including TSG101, CD81, and CD63 (Figure 2(e)). In addition, H9c2 cells was incubated with pKH26-labeled small extracellular vesicles for 24 h to assess whether MSC-derived small extracellular vesicles could be taken up by H9c2 cells. Microscopic analysis showed that MSC-derived small extracellular vesicles could be absorbed by H9c2 cells (Figure 2(f)).

EMPA-sEV Protects H9c2 Cells by Inhibiting Apoptosis.
A large number of studies have shown that MSC-derived small extracellular vesicles can protect cardiomyocytes from ischemia and hypoxia by inhibiting apoptosis [22]. Through the above experiments, we confirmed that EMPA can improve the activity of MSCs and has no adverse effect on the secretion of small extracellular vesicles by MSCs. To verify whether EMPA-sEV enhances the protective effect on cardiomyocytes, H9c2 cells were induced by H 2 O 2 (120 μM) under serum deprivation conditions. EMPA-sEV, MSC-sEV, or PBS were then treated with the apoptotic cardiomyocytes for 12 h. By caspase-3/7 staining, we found that the caspase-3/7-positive cells were significantly reduced in the EMPA-sEV group compared with the PBS group and MSC-sEV group (Figures 3(a) and 3(b)). PI staining showed that the PI-positive cells in the EMPA-sEV group were also significantly lower than those in the other two groups (Figures 3(c) and 3(d)). In addition, compared with the other groups, the expression of proapoptotic proteins (BAX) was significantly decreased and the expression of (f) Fluorescence microscope analysis showed that small extracellular vesicles labeled with red fluorescent dye PKH26 were endocytosed by H9c2 cells after 12 h incubation (scale = 100 μm, 20 μm) (n = 2). Data are expressed as mean ± SEM. * P < 0:05, * * P < 0:01, * * * P < 0:001, and * * * * P < 0:0001.

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Oxidative Medicine and Cellular Longevity antiapoptotic proteins (Bcl-2) was significantly increased in the EMPA-sEV group (Figure 3(e)). These results suggest that EMPA-pretreated MSC-derived small extracellular vesicles can better protect cardiomyocytes by inhibiting apoptosis compared with normal small extracellular vesicles from MSCs.

EMPA-sEV Promotes Cardiomyocyte Survival and
Angiogenesis in MI Rats. To demonstrate whether EMPA-sEV has a more significant cardioprotective effect in vivo, we established a SD rat model of myocardial infarction by ligation of the left anterior descending coronary artery. To observe the effect of EMPA-sEV on cardiac function, cardiac ultrasound was performed on rats in each group at 23 and 36 days after MI surgery. Treatment with small extracellular vesicles significantly improved cardiac function compared with the MI group. At the same time, the EMPA-sEV group showed a greater improvement in cardiac function (Figures 4(a)-4(c)). In addition, Masson staining showed that small extracellular vesicles therapy reduced infarct size. EMPA-sEV was superior to MSC-sEV in reducing infarct size (Figure 4(d)). Sirius red staining also showed that EMPA-sEV inhibited myocardial fibrosis better than MSC-sEV ( Figure 5(b)). As can be seen from Figure 5(a), there are obvious inflammatory cell infiltration in the post-MI infarct area and the infarct border area. However, the small extracellular vesicle treatment groups effectively reduced inflammatory cell infiltration, and EMPA-sEV significantly improved inflammatory cell infiltration in the infarct area and infarct margin area. In addition, the heart tissue sections were stained with immunofluorescence and immunohistochemistry. Compared with the MI group and MSC-sEV group, the EMPA-sEV group had lower TUNEL-positive rate (Figures 6(a) and 6(c)), higher CD31-positive rate (Figures 6(a) and 6(d)), and higher Bcl-2-positive rate (Figures 6(b) and 6(e)). These data suggest that EMPA-sEV could inhibit myocardial cell apoptosis and promote angiogenesis better than MSC-sEV (Figures 6(a)-6(e)). In summary, small extracellular vesicle therapy can effectively improve cardiac function after infarction by inhibiting myocardial apoptosis, myocardial fibrosis, and promoting angiogenesis. Furthermore, EMPA-pretreated MSC-derived small extracellular vesicles have more significant inhibitory effects on cardiomyocyte apoptosis and myocardial fibrosis, . Data are expressed as mean ± SEM. * P < 0:05, * * P < 0:01, * * * P < 0:001, and * * * * P < 0:0001. 7 Oxidative Medicine and Cellular Longevity as well as more significant angiogenesis promotion and cardiac function improvement.

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Oxidative Medicine and Cellular Longevity Next, whether miR-214-3p mediates the cardioprotective effect of EMPA-pretreated MSC-derived small extracellular vesicles was further verified. MSCs were transfected with NC mimics and miR-214 mimics, respectively. Then, small extracellular vesicles were extracted and cocultured with H9c2 cells. RT-qPCR verified that mR-214-3p expression was increased in H9c2 cells compared with the control groups (Figure 8(a)). Compared with the negative control group, the expression of antiapoptotic protein (Bcl-2) was increased and the expression of proapoptotic protein (BAX) was decreased in the miR-214 mimics group under ischemia and hypoxia (Figures 8(b) and 8(c)). These results suggest that miR-214-3p can mediate the antiapoptotic effect of EMPA-pretreated MSC-derived small extracellular vesicles. Based on the results of miRNA target gene KEGG analysis, we focused on the AKT signaling pathway. Studies have confirmed that the AKT signaling pathway plays a key role in inhibiting myocardial apoptosis. To verify whether miR-214-3p activates the AKT signaling pathway, we observed the expression levels of the AKT signaling pathway-related proteins. As can be seen from Figures 8(b) and 8(c), p-AKT expression was significantly increased in the miR-214 mimics group.

Discussion
The current study has several major findings. First, EMPA can enhance cell viability and promote proliferation and migration of MSCs. EMPA also can inhibit the senescence of MSCs. Second, compared with MSC-sEV, EMPA-sEV has a more significant protective effect on cardiac function, including promoting angiogenesis and inhibiting fibrosis. Finally, the cardioprotective effect of EMPA-sEV is mediated, at least in part, by miR-214-3p via the AKT signaling pathway.
In recent years, research on MSC transplantation has made great progress and become a potential treatment for ischemic heart disease [27]. MSCs are a pluripotent stem cell population that can be isolated from the bone marrow, fat, umbilical cord blood, etc. [28]. MSCs are characterized by multidirectional differentiation potential and low immunogenicity [29,30]. Studies have shown that intravenous injection of MSCs can be tolerated, while repeated intracardial or coronary cell delivery cannot [31]. Nevertheless, there are still many security issues with MSC transplantation [32,33]. The aging of MSCs reduces their cardioprotective effect [34]. The complex microenvironment and poor localization control of infarcted hearts after transplantation also limit the therapeutic effect of MSC transplantation [35]. To improve the therapeutic effect and survival of MSC transplantation in the ischemic and hypoxic microenvironment, many studies have been reported. These include genetic engineering and pretreatment of pharmacological . Data are expressed as mean ± SEM. * P < 0:05, * * P < 0:01, * * * P < 0:001, and * * * * P < 0:0001.

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Oxidative Medicine and Cellular Longevity compounds. A recent study demonstrated that high expression of miR-221-3p can enhance the cardioprotective effects of aging MSCs after myocardial infarction through the PTEN/AKT pathway, including promoting angiogenesis and inhibiting apoptosis [20]. Another study showed that the nonadherent culture of MSCs promoted angiogenesis and reduced cardiac remodeling after MSC transplantation by increasing the secretion of hydrolytic growth factor A (VEGFA) [36]. In this study, MSCs were pretreated with SGLT2i (EMPA). As a new hypoglycemic agent, SGLT2i has a cardiovascular protective effect independent of the hypoglycemic effect [37]. The cardioprotective effect of SGLT2i may be mediated by its ability to reduce cardiac inflammation, oxidative stress, apoptosis, and ion imbalance [38][39][40][41]. EMPA pretreatment increased the cell viability, proliferation, and migration of MSC and decreased the senescence of MSC. Further studies will clarify whether the beneficial effect of EMPA on MSCs can partially explain the cardiovascular protective mechanism of SGLT2i.

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Oxidative Medicine and Cellular Longevity vesicles can inhibit autophagy of ischemic cardiomyocytes and promote endothelial micro-angiogenesis [45]. Wei et al. reported that the overexpression of miRNA-181a in MSC-derived small extracellular vesicles affects the inflammatory response after myocardial ischemia-reperfusion injury [46]. Although these genetic approaches can directly alter the expression of cytokines or genes related to cardiac protection in MSC-derived small extracellular vesicles, they are not currently feasible in clinical practice. In contrast, we pretreated MSCs with EMPA with higher clinical feasibility. Our study found that EMPA-sEV inhibited H9c2 cells apoptosis better than MSC-sEV. Compared with MSC-sEV, EMPA-sEV had a better protective effect on cardiac function, promoting angiogenesis and inhibiting fibrosis. It (d) Simple mechanism diagram of this study. Data are expressed as mean ± SEM. * P < 0:05, * * P < 0:01, * * * P < 0:001, and * * * * P < 0:0001.
13 Oxidative Medicine and Cellular Longevity is well known that small extracellular vesicles perform biological functions by containing proteins, mRNAs, miR-NAs, etc [47]. In recent years, there has been increasing evidence that small extracellular vesicles' miRNAs mediate many biological functions of small extracellular vesicles. miRNAs including miR-22, miR-199a, and miR-214 play a key role in antiapoptosis effects [22]. In this study, the miRNA expression profile in EMPA-sEV showed that miR-214-3p in EMPA-sEV was significantly higher than that in MSC-sEV. In addition, the expression level of miR-214-3p in H9c2 cells was also increased after treatment with EMPA-sEV.
miR-214-3p has been reported to have cardioprotective effects. miR-214-3p plays an antiapoptotic role by regulating sodium/calcium recovery 1, cyclophilin D, and Bcl-2 like protein 11 [48]. Small extracellular vesicles derived from miR-214-enriched MSCs reduce cardiac stem cell death by inhibiting reactive oxygen species production. In contrast, miR-214 inhibitors attenuate these effects [49,50]. Based on these and other observations, we propose that miR-214-3p mediates the role of EMPA-sEV in cardiac protection. By overexpressing miR-214-3p in MSCs, the same antiapoptotic effect as that of EMPA-sEV was obtained. These results support the important role of miR-214-3p in anti-apoptosis in EMPA-sEV-mediated cardiovascular protection. In addition, we found that miR-214-3p promotes the phosphorylation of AKT in H9c2 cells, suggesting a possible molecular mechanism of miR-214-3p's role in cardiac protection. It has been reported that activation of the AKT signaling pathway is a key target for cardiac protection [51]. Current evidence suggests that miR-214-3p exerts cardioprotective effects by enhancing the AKT signaling pathway in cardiomyocytes. However, further studies are needed to elucidate the downstream mechanisms of the cardioprotective effects of EMPA-sEV mediated by miR-214-3p in physiology and pathophysiology.
4.1. Limitation. The current study has some limitations. Firstly, H9c2 cells do not represent cardiomyocytes well. Cardiac primary cardiomyocytes are a good source for in vitro research. Secondly, our study suggests that the cardioprotective effects of EMPA-sEV were at least partly mediated through miR-214. Other miRNAs, proteins, lipids, and mRNAs which were also contained in small extracellular vesicles may also be functionally involved with the cardioprotective effects. However, these molecules have not been further investigated in this study. Thirdly, EMPA-sEV is at least partially protected by miR-214-3p through the AKT signaling pathway, but its specific mechanism has not been elucidated. Finally, although EMPA-sEV has a more significant therapeutic effect compared to MSC-sEV, further preclinical studies are needed to validate its efficacy and safety.

Conclusion
EMPA pretreatments promoted the effect of MSC-derived small extracellular vesicles on inhibiting myocardial apoptosis, enhancing angiogenesis, and improving cardiac function after myocardial infarction. MiR-214-3p at least partly medi-ated the cardioprotective effects of EMPA-sEV via activating the AKT signaling pathway.

Data Availability
The datasets and materials used in the study are available from the corresponding author.